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| United States Patent |
5,279,823
|
|
Frenz
, et al.
|
January 18, 1994
|
Purified forms of DNASE
Abstract
The present invention provides the identification and characterization of
two components of a recombinant preparation of DNase. These components are
the purified deamidated and non-deamidated human DNases. Taught herein are
the separation of these components and the use of the non-deamidated
species as a pharmaceutical per se, and in particular in compositions
wherein the species is disclosed within a plastic vial, for use in
administering to patients suffering from pulmonary distress.
| Inventors:
|
Frenz; John (Millbrae, CA);
Shire; Steven J. (Belmont, CA);
Sliwkowski; Mary B. (San Carlos, CA)
|
| Assignee:
|
Genentech, Inc. (South San Francisco, CA)
|
| Appl. No.:
|
895300 |
| Filed:
|
June 8, 1992 |
| Current U.S. Class: |
424/94.61; 435/199; 435/814; 435/815 |
| Intern'l Class: |
C12N 009/22; A61K 037/54 |
| Field of Search: |
435/199,814,815
424/94.61,499
|
References Cited
U.S. Patent Documents
| 4065355 | Dec., 1977 | Khouw et al. | 435/199.
|
Other References
Liao et al., J. Biol. Chem., 248 (4): 1489-1495 (1973).
Lovrenco et al., Arch. Intern. Med., 142:2299-2308 (1982).
Shak et al., PNAS USA, 87:9188-9192 (1990).
Hubbard et al., N. Eng. J. Med., 326 (12):812-815 (1992).
Markey, FEBS, 167 (1):155-159 (1984).
Nefsky et al., Eur. J. Biochem., 179:215-219 (1989).
Eipper et al., Ann. Rev. Physiol., 50:333-344 (1988).
Kassiakoff, Science, 240:191-194 (1988).
Bradbury et al., TIBS, 16:112-115 (1991).
Wright, Protein Engineering, 4(3):283-294 (1991).
|
Primary Examiner: Wax; Robert A.
Assistant Examiner: Prouty; Rebecca
Attorney, Agent or Firm: Johnston; Sean A.
Claims
What is claimed is:
1. Purified deamidated human DNase.
2. Purified non-deamidated human DNase.
3. A pharmaceutical composition consisting of deamidated human DNase as the
active principle and optionally a pharmaceutically acceptable excipient.
4. A pharmaceutical composition consisting of non-deamidated human DNase as
the active principle and optionally a pharmaceutically acceptable
excipient.
5. A pharmaceutical composition according to claim 4 wherein the excipient
is sterile water.
6. A pharmaceutical composition according to claim 4 wherein the excipient
is a sterile unbuffered aqueous solution at about pH 4.5-6.8.
7. A pharmaceutical composition according to claim 4 wherein said
composition is in an aerosol form.
8. A pharmaceutical composition comprising non-deamidated human DNase that
is substantially free of deamidated human DNase that is substantially free
of deamidated human DNase.
Description
RELATED PATENT APPLICATIONS
The present application is related in subject matter to the disclosure
contained in U.S. patent application Ser. No. 07/448,038, filed 8 Dec.
1989 and U.S. patent application Ser. No. 07/289,958, filed 23 Dec. 1988.
The subject matter of those applications is in the public domain by virtue
of the publication, inter alia, of the PCT counterpart on or about 12 Jul.
1990 as International Patent Application Publication No. WO 90/07572. The
content of these prior applications is hereby expressly incorporated by
reference herein.
FIELD OF THE INVENTION
The present invention is related to results obtained from research on
deoxyribonuclease (DNase), a phosphodiesterase that is capable of
hydrolyzing polydeoxyribonucleic acid. It relates generally to the
separation of several forms of said DNase, to these forms per se, to
pharmaceutical compositions by which their utility can be exploited
clinically, and to methods of using these DNases and compositions thereof.
BACKGROUND OF THE INVENTION
DNase is a phosphodiesterase capable of hydrolyzing polydeoxyribonucleic
acid. DNase has been purified from various species to various degrees. The
complete amino acid sequence for a mammalian DNase was first made
available in 1973. See e.g., Liao, et al., J. Biol. Chem. 248:1489 (1973).
DNase has a number of known utilities and has been used for therapeutic
purposes. Its principal therapeutic use has been to reduce the
viscoelasticity of pulmonary secretions in such diseases as pneumonia and
cystic fibrosis, thereby aiding in the clearing of respiratory airways.
See e.g., Lourenco, et al., Arch. Intern. Med. 142:2299 (1982); Shak, et
al., Proc. Nat. Acad. Sci. 87:9188 (1990); Hubbard, et al., New Engl. J.
Med. 326:812 (1992).
DNA encoding human DNase I has been isolated and sequenced and that DNA has
been expressed in recombinant host cells, thereby enabling the production
of human DNase in commercially useful quantities. See e.g., Shak, et al.,
Proc. Nat. Acad. Sci. 87:9188-9192 (1990). Recombinant human DNase
(rhDNase) has been found to be useful clinically, especially in purified
form such that the DNase is free from proteases and other proteins with
which it is ordinarily associated in nature. See e.g., Hubbard, et al.,
New Engl. J. Med. 326:812 (1992).
The means and methods by which human DNase can be obtained in
pharmaceutically effective form is described in the patent applications
cited above. Various specific methods for the purification of DNase are
known in the art. See e.g., Khouw, et al., U.S. Pat. No. 4,065,355 (issued
27 Dec. 1977); Markey, FEBS Letters 167:155 (1984); Nefsky, et al., Eur.
J. Biochem. 179:215 (1989).
Although it was not appreciated at the time the above-referenced patent
applications were filed, the DNase product obtained from cultures of
recombinant host cells typically comprises a mixture of deamidated and
non-deamidated forms of DNase. The existence of deamidated forms of DNase
remained unappreciated notwithstanding that the phenomenon of deamidation
of asparagine and glutamine residues in some proteins is known. See e.g.,
Eipper et al., Ann. Rev. Physiol. 50:333 (1988); Kossiakoff, Science
240:191 (1988); Bradbury et al., Trends in Biochem. Sci. 16:112 (1991);
and Wright, Protein Engineering 4:283 (1991);
The present invention is predicated upon the previously unappreciated fact
that recombinant human DNase may exist as a mixture of deamidated and
non-deamidated forms. Using the methods of the present invention, it has
been found that deamidated human DNase is less active enzymatically than
non-deamidated human DNase. Thus, the presence of the deamidated DNase and
non-deamidated DNase together in a mixture, and the potential for further
deamidation occurring, such as has been found to occur upon in vitro
storage of preparations of human DNase, may complicate efforts to provide
consistent uniformity in a DNase product being administered clinically.
Therefore, as the existence and characteristics of deamidated DNase were
not known prior to the present invention, the methods for identifying
deamidated DNase and separating it from preparations of DNase in which it
may be found were unobvious at the time this invention was made.
SUMMARY OF THE INVENTION
The present invention is directed to processes for separating the
deamidated and non-deamidated human DNase forms from a mixture thereof.
This process in preferred embodiments comprises subjecting the mixture to
chromatography using a resin, or other support medium, having bound
thereto a cationic polymer such as heparin or a non-hydrolyzable
deoxyribonucleic acid (DNA) analog, or chromatography using a so-called
tentacle cation exchange resin. The present invention also is directed to
the use of those chromatographic methods with non-human DNases, such as
bovine DNase.
The present invention also is directed to deamidated human DNase as a
purified product, substantially free of non-deamidated human DNase.
The present invention also is directed to non-deamidated human DNase as a
purified product, substantially free of deamidated human DNase. It has
been found herein that purified non-deamidated human DNase is fully
enzymatically active as compared with deamidated human DNase.
The present invention also is directed to pharmaceutical compositions
consisting of either purified deamidated human DNase or purified
non-deamidated human DNase as the active principle, optionally together
with a pharmaceutically acceptable excipient.
The present invention also is directed to a method comprising administering
a therapeutically effective amount of purified deamidated human DNase or
purified non-deamidated human DNase for the treatment of a patient, for
example those having an accumulation of viscous, DNA-containing material.
The administration of such purified DNases preferably is effected by
direct inhalation into the lungs.
The present invention is particularly directed to a method of treating a
patient having a pulmonary disease such as chronic bronchitis, cystic
fibrosis, or emphysema, that comprises administering a therapeutically
effective amount of purified non-deamidated human DNase, preferably
directly into the airway passages.
The present invention also is directed to pharmaceutical compositions
comprising non-deamidated human DNase that are disposed within a plastic
vial, optionally in the presence of a pharmaceutically acceptable
excipient.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1B depict the amino acid (SEQ. ID. NO. 1) and DNA sequences (SEQ.
ID. NO. 2) of human DNase I. The native signal sequence is underlined, the
potential initiation codons are circled, and the mature sequence is
bracketed.
FIG. 2 depicts the correlation between enzymatic activity and extent of
deamidation of samples of human DNase. Specific activity was determined by
normalizing the DNase activity as determined by a methyl green (MG) assay
(in concentration units relative to a standard curve) to the DNase
concentration measured by an enzyme-linked immunoabsorbent assay (ELISA).
Percent deamidation was determined by tryptic mapping. "Day of Harvest"
samples of human DNase were purified from a culture of recombinant Chinese
hamster ovary (CHO) cells expressing DNA encoding human DNase I. Such
samples were taken at 3, 5, 7, 9, 11, 13, and 20 days after the culture
was started. "High pH" samples were day 13 samples of purified DNase that
were incubated in vitro for two days at pH 8 at 37.degree.. "Stability"
samples were day 13 samples of purified DNase that were stored in vitro at
5.degree., 25.degree., or 37.degree. C. for various periods of time.
FIG. 3 is an example of a tryptic map of DNase employed for determination
of the extent of deamidation. The sample shown here is 65% deamidated
DNase. "mAU" indicates milli-absorbance units at 214 nM.
FIG. 4 is a schematic representation of the deamidation of the asparagine
residue at amino acid position 74 (Asn-74) in native human DNase.
Deamidation converts the Asn-74 to either an aspartic acid (Asp) or an
iso-aspartate (iso-Asp) residue. Each of the three forms of DNase yields,
on digestion with trypsin, a pair of peptides that indicates the identity
of the particular form of DNase.
FIG. 5 is a chromatogram of a human DNase sample fractionated on a tentacle
cation exchange (TCX) column. The sample shown is 67% deamidated DNase.
FIG. 6 shows tryptic maps of the two peak fractions from the TCX separation
shown in FIG. 5. The absence of tryptic peptide T6-7 from the map of the
Peak 2 digest indicates the absence of deamidated DNase.
FIG. 7 shows chromatograms of several human DNase samples fractionated on a
TCX column. The sample designated "M1-28 STD." is a preparation of human
DNase obtained from a culture of Chinese hamster ovary (CHO) cells
transformed with DNA encoding native human DNase I. The sample designated
"DNase ASP Mutant" is DNase having an aspartic acid residue (rather than
an asparagine residue) at amino acid position 74, and which thus has the
same amino acid sequence as the Asp form of deamidated DNase shown in FIG.
4. The DNase ASP Mutant was obtained from a culture of cells transformed
with DNA encoding that mutant form of human DNase. The DNA encoding the
DNase ASP Mutant was prepared by site-directed mutagenesis of DNA encoding
native human DNase. Comparison of the chromatograms shows that one of the
forms of human DNase in the M1-28 STD. elutes from the TCX column at the
same position as the DNase Asp Mutant.
FIG. 8 shows chromatograms of several human DNase samples fractionated on a
TSK-Heparin column (Toso Haas, Montgomeryville, Pa.). The sample
designated "12K #8" is a preparation of human DNase obtained from a
culture of Chinese hamster ovary (CHO) cells transformed with DNA encoding
native human DNase I. The sample designated "Deamidated Standard" is
purified deamidated human DNase. The sample designated "Non-deamidated
standard" refers to purified non-deamidated human DNase. Purified
deamidated human DNase and purified non-deamidated human DNase were
prepared by TCX chromatography.
FIG. 9 shows chromatograms of several human DNase samples fractionated on
an immobilized DNA analog column. The sample designated "M1-28" is a
preparation of human DNase obtained from a culture of Chinese hamster
ovary (CHO) cells transformed with DNA encoding native human DNase I. The
sample designated "Deamidated Standard" is purified deamidated human
DNase. The sample designated "Non-deamidated standard" refers to purified
non-deamidated human DNase. Purified deamidated human DNase and purified
non-deamidated human DNase was prepared by TCX chromatography. The sample
designated "DNase ASP Mutant" is DNase having an aspartic acid residue
(rather than an asparagine residue) at amino acid position 74.
DETAILED DESCRIPTION
A. Definitions
By the term "human DNase" herein is meant a polypeptide having the amino
acid sequence of human mature DNase I set forth in FIGS. 1A-1B as well as
amino acid sequence variants thereof (including allelic variants) that are
enzymatically active in hydrolyzing DNA. Thus, the term "human DNase"
herein denotes a broad definition of those materials disclosed and
prepared in the patent applications described above.
The term "human DNase" necessarily embraces native mature human DNase
having an asparagine (Asn) residue at amino acid position 74 of the
polypeptide. That asparagine has been found herein to be susceptible to
deamidation, which deamidation may produce a mixture of deamidated and
non-deamidated forms of human DNase. Instead of the Asn residue at amino
acid position 74, deamidated DNase has an aspartic acid (Asp) or an
iso-aspartate (iso-Asp) residue (see FIG. 4).
The term "deamidated human DNase" as used herein thus means human DNase
that is deamidated at the asparagine residue that occurs at position 74 in
the amino acid sequence of native mature human DNase. It has been found
that deamidated human DNase may arise during the production of human DNase
by recombinant means, and may be found in preparations of human DNase
obtained from recombinant host cells. Additionally, deamidated human DNase
may arise upon in vitro storage of non-deamidated human DNase.
Although the asparagine residue at amino acid position 7 in the amino acid
sequence of native mature human DNase also may be deamidated (in addition
to the asparagine residue at amino acid position 74), such doubly
deamidated DNase has been found to be enzymatically inactive.
The term "mixture" as used herein in reference to preparations of human
DNase means the presence of both deamidated and non-deamidated forms of
human DNase. It has been found, for example, that in preparations of human
DNase obtained from recombinant expression, as much as about 50% to 80% or
more of the human DNase is deamidated.
The term "purified deamidated human DNase" as used herein means deamidated
human DNase that is substantially free of non-deamidated human DNase. In
other words, non-deamidated human DNase will comprise less than about 10%,
preferably less than about 5%, and most preferably less than about 1% by
weight of the total DNase in the purified deamidated human DNase
composition.
The term "purified non-deamidated human DNase" as used herein means
non-deamidated human DNase that is substantially free of deamidated human
DNase. In other words, deamidated human DNase will comprise less than
about 25%, preferably less than about 5%, and most preferably less than
about 1% by weight of the total DNase in the purified non-deamidated human
DNase composition.
By the term "excipient" herein is meant a pharmaceutically acceptable
material that is employed together with DNase for the proper and
successful administration of the DNase to a patient. Suitable excipients
are well known in the art, and are described, for example, in the
Physicians Desk Reference, the Merck Index, and Remington's Pharmaceutical
Sciences.
A preferred formulation for human DNase is a buffered or unbuffered aqueous
solution, and preferably is an isotonic salt solution such as 150 mM
sodium chloride containing 1.0 mM calcium chloride at pH 7. These
solutions are particularly adaptable for use in commercially-available
nebulizers including jet nebulizers and ultrasonic nebulizers useful for
administration, for example directly into the airways or lungs of an
affected patient. Reference is made to the above-identified patent
applications for further detail concerning how human DNase can be
formulated and administered for effective use.
By the term "therapeutically effective amount" herein, is meant dosages of
from about 1 .mu.g to about 100 mg of human DNase per kilogram of body
weight of the patient, administered within pharmaceutical compositions, as
described herein. The therapeutically effective amount of human DNase will
depend, for example, upon the therapeutic objectives, the route of
administration, and the condition of the patient. Accordingly, it will be
necessary for the therapist to titer the dosage and modify the route of
administration as required to obtain the optimal therapeutic effect. In
view of the differences in enzymatic activity between deamidated and
non-deamidated DNases described herein, it may be that the amount of
purified non-deamidated DNase required to achieve a therapeutic effect
will be less than the amount of purified deamidated human DNase or a
mixture of the two forms necessary to achieve the same effect under the
same conditions.
The purified DNases hereof, particularly the non-deamidated form, are
employed for enzymatic alteration of the viscoelasticity of mucous. Such
purified human DNases are particularly useful for the treatment of
patients with pulmonary disease who have abnormal viscous, purulent
secretions and conditions such as acute or chronic bronchial pulmonary
disease, including infectious pneumonia, bronchitis or tracheobronchitis,
bronchiectasis, cystic fibrosis, asthma, tuberculosis, and fungal
infections. For such therapies, a solution or finely divided dry
preparation of purified deamidated human DNase or purified non-deamidated
human DNase is instilled in conventional fashion into the bronchi, for
example by aerosolization.
B. Preferred Embodiments
After the successful cloning and expression of human DNase in recombinant
host cells, it was discovered after substantial research that the DNase
product obtained from such recombinant expression typically existed as a
mixture of as then yet undefined components. In particular, isoelectric
focusing (IEF) analysis of human DNase purified from cultures of
recombinant Chinese hamster ovary (CHO) cells revealed a complex pattern
of DNase species. The various DNase species were determined to result from
several post-translational modifications of the DNase, including
deamidation.
Two assays were used to determine the presence and extent of deamidated
DNase in such preparations. One method involved tryptic digestion of the
starting preparation of DNase and analysis of the resulting peptides by
reverse phase HPLC. In this method, the amount of deamidated DNase in the
starting preparation was determined by measuring the quantities of six
deamidation-indicating tryptic peptides.
The other method involved chromatography of the starting preparation of
DNase on a tentacle cation exchange (TCX) column. It was discovered that
the TCX column is capable of resolving deamidated human DNase and
non-deamidated human DNase, such that each form of DNase could be
effectively separated from the other, and obtained in purified form. In
this method, the amount of deamidated and non-deamidated DNase in the
starting preparation was determined by measuring on chromatograms the peak
areas corresponding to the separated forms of DNase.
Although these two methods are about equally effective in determining and
quantitating deamidated DNase, the TCX method is especially efficient,
requiring far less time and labor than the other method. Moreover, TCX
chromatography provides a means for separating deamidated and
non-deamidated forms of DNase, whereas conventional cation exchange resins
and various other chromatography resins that were analyzed were not
capable of such separation.
The general principles of TCX chromatography have been described, for
example, by Miller, J. Chromatography 510:133 (1990); Janzen et al., J.
Chromatography 522:77 (1990); and Hearn et al., J. Chromatography 548:117
(1991). Without limiting the invention to any particular mechanism or
theory of operation, it is believed that the Asn-74 residue in human DNase
that is susceptible to deamidation is located within the DNA-binding
groove of the enzyme, by analogy to the known crystal structure of bovine
DNase. The DNA-binding groove contains basic amino acid residues (in order
to bind DNA) and this groove apparently is accessible to the ligands of
the tentacle cation exchange resin but not to the much shorter ligands of
conventional cation exchange resins. Presumably the ligands of the
tentacle cation exchange resin mimic natural nucleic acid substrates.
Therefore, it is expected that tentacle action exchange chromatography
will be useful for the purification of other nucleases, such as
ribonuclease (RNase) or restriction endonucleases, as well as DNA binding
proteins.
Alternatively, the separation of deamidated and non-deamidated forms of
DNase may be accomplished by chromatography using a resin or other support
matrix containing covalently bound cationic polymers such as heparin or a
synthetic non-hydrolyzable DNA analog. Immobilized heparin chromatography
columns are commercially available (for example, from Toso Haas Co.,
Montgomeryville, Pa.). Non-hydrolyzable DNA analogs have been described,
for example, by Spitzer et al., Nuc. Acid. Res. 16:11691 (1988). An
immobilized non-hydrolyzable DNA analog column is conveniently prepared by
synthesizing such a DNA analog with an amino acid group at the 3'-end of
one or both of its complementary strands. The amino group is then
available for coupling to an epoxy-activated column, as described, for
example, in literature published by Rainin Biochemical LC Products
(Woburn, Mass.).
Following the successful separation of deamidated and non-deamidated human
DNases according to the methods of the present invention, it was found
that deamidated human DNase has diminished enzymatic activity as compared
to non-deamidated human DNase, as determined by a methyl green (MG) assay.
Kurnick, Arch. Biochem. 29:41 (1950). It was found that deamidated human
DNase exhibits just over half of the enzymatic activity of non-deamidated
human DNase. Thus, by combining the purified deamidated DNases and the
purified non-deamidated DNase of the present invention in various
proportions, it is possible to prepare pharmaceutical compositions of
human DNase having any desired specific activity in the range between the
specific activities of the individual components, as may be optimal for
treating particular disorders.
The following examples are offered by way of illustration only and are not
intended to limit the invention in any manner. All patent and literature
references cited throughout the specification are expressly incorporated
herein.
C. Examples
1. Tryptic Mapping
The procedure used for tryptic mapping of human DNase is summarized as
follows:
Step 1. Bring concentration of 1 mg sample of DNase to 4 mg/ml by
concentration on Amicon Centricon-10 device or by dilution with excipient.
Final volume: 250 .mu.l.
Step 2. Add 250 .mu.l of pretreatment buffer (40 mM BisTris, 10 mM EGTA, pH
6.0) to sample. Incubate 1 hour at 37.degree..
Step 3. Buffer exchange sample into digest buffer (100 mM Tris, pH 8) using
Pharmacia NAP-5 column. Final volume: 1 ml.
Step 4. Add 10 .mu.l trypsin solution (1 mg/ml trypsin, 1 mM HCl) to sample
and incubate 2 hours at 37.degree..
Step 5. Add second 10 .mu.l aliquot of trypsin solution to sample and
incubate additional 2 hours at 37.degree..
Step 6. Stop digestion by addition of 6 .mu.l trifluoroacetic acid (TFA).
Store samples at or below 5.degree. until chromatographed.
Step 7. Separate the peptide mixture by HPLC under the following
conditions:
Column: Nucleosil C18, 5 .mu.m, 100 .ANG., 2.0.times.150 mm (Alltech, Co.,
Deerfield, Ill.).
Column temperature: 40.degree.
Eluent A: 0.12% TFA in water.
Eluent B: 0.10% TFA in acetonitrile.
Gradient profile:
______________________________________
Time (min) % A % B
______________________________________
0 100 0
5 100 0
65 40 60
69 5 95
70 5 95
______________________________________
Flow rate: 0.25 ml/min. Sample
injection volume: 250 .mu.l.
Post-run column reequilibration time at
100% A: 20 min.
Autosampler compartment temperature:
5.degree..
Detection: Absorbance at 214 and 280 nm.
Step 8. Identify T7, (D)T7, T7-8, (D)T7-8, T6-7-8, and T6-7 tryptic
peptides by retention time comparison with standard.
Step 9. Integrate chromatogram obtained at 280 nm. Check quality of
integration by inspection of baseline and separation of closely eluting
peaks. Special attention must be paid to the early-eluting T7 and (D)T7
peptides that may not be well-resolved.
Step 10. Normalize peak areas of the six reporter peptides to tyrosine
content. Peptides T7, (D)T7, T7-8, and (D)T7-8 each contain a single Tyr
residue, while T6-7-8 and T6-7 contain three Tyr residues. Calculate the
proportion of deamidated species based on the normalized peak areas of
(D)T7, (D)T7-8, T6-7-8, and T6-7 relative to the total normalized peak
areas of the six peptides.
One milligram of DNase in a volume of 250 .mu.l is required in order to
accurately carry out the tryptic mapping method for determination of
deamidated DNase according to the procedure outlined above. Hence, the
initial sample preparation for this method requires either concentration
or dilution of the sample to achieve that result. DNase in the presence of
calcium is highly resistant to proteases, including trypsin. Therefore the
next step in the procedure is to partially remove calcium ions by
treatment with [ethylene bis(oxyethylenenitrilo)] tetraacetic acid (EGTA).
Over-treatment with EGTA can denature and aggregate DNase, so this step
must be performed with care. The EGTA-treated sample in a volume of 0.5 ml
is then exchanged into 1 ml of the digest buffer, trypsin added, and the
sample incubated at 37.degree. for two hours. A second aliquot of trypsin
is then added and the sample incubated an additional two hours. Digestion
is stopped by acidification, and the sample is either stored for later
analysis or loaded on the HPLC column directly.
250 .mu.l (250 .mu.g) of the peptide mixture resulting from the tryptic
digestion is separated on a reversed phase HPLC column according to the
conditions outlined above. A typical tryptic map of human DNase is shown
in FIG. 3. HPLC was performed with a Hewlett-Packard model 1090M HPLC. The
column effluent was monitored simultaneously at 214 and 280 nm by the
diode array detector that is a feature of this instrument. Since the early
portion of the peptide map is critical to the quantitation of deamidated
DNase, as described below, other instruments with larger gradient delay
and other extra-column volumes may not be suited to this analysis. Each
analysis by this procedure requires 70 minutes for the gradient separation
and 20 minutes to re-equilibrate the column for a total HPLC turnaround
time of 90 minutes. The rationale and approach to peak integration for
determination of deamidated DNase in a sample are described below.
Deamidation of human DNase occurs at least at the asparagine residue that
is present at amino acid position 74 (Asn-74) in native mature human
DNase. Asn-74 is on the C-terminal side of a tryptic cleavage site at the
arginine residue at amino acid position 73 (Arg-73), as seen in the list
of expected tryptic peptides of human DNase shown in Table I.
TABLE I
__________________________________________________________________________
PEPTIDES EXPECTED TO BE PRODUCED UPON
DIGESTION OF NATIVE MATURE HUMAN DNASE
WITH TRYPSIN.
ID Residues
Amino Acid Sequence of Peptide
__________________________________________________________________________
T1 1-2 LK
T2 3-15
IAAFNIQTFGETK (SEQ. ID. NO. 3)
T3 16-31
MSNATLVSYIVQILSR (SEQ. ID. NO. 4)
T4 32-41
YDIALVQEVR (SEQ. ID. NO. 5)
T5 42-50
DSHLTAVGK (SEQ. ID. NO. 6)
T6 51-73
LLDNLNQDAPDTYHYVVSEPLGR (SEQ. ID. NO. 7)
T7 74-77
NSYK (SEQ. ID. NO. 8)
T8 78-79
ER
T9 80-111
YLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNR (SEQ. ID. NO. 9)
T10
112-117
EPAIVR (SEQ. ID. NO. 10)
T11
118-121
FFSR (SEQ. ID. NO. 11)
T12
122-126
FTEVR (SEQ. ID. NO. 12)
T13
127-157
EFAIVPLHAAPGDAVAEIDALYDVYLDVQEK (SEQ. ID. NO. 13)
T14
158-185
WGLEDVMLMGDFNAGCSYVRPSQWSSIR (SEQ. ID. NO. 14)
T15
186-213
LWTSPTFQWLIPDSADTTATPTHCAYDR (SEQ. ID. NO. 15)
T16
214-222
IVVAGMLLR (SEQ. ID. NO. 16)
T17
223-260
GAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEMLK
(SEQ. ID. NO. 17)
__________________________________________________________________________
Instead of the Asn (single letter designation "N") residue at residue 74 in
native, non-deamidated human DNase, deamidated human DNase has either an
Asp or iso-Asp residue, as shown in FIG. 4. Iso-Asp is an isomeric,
beta-amino acid form of aspartic acid. The peptide bond between Arg-73 and
iso-Asp is resistant to cleavage by trypsin, so deamidated human DNase
yields a characteristic tryptic peptide containing residues 51-77 and
called T6-7 since it is the conjoined peptides T6 and T7. Under conditions
employed for tryptic mapping, the Arg-73-Asn-74 peptide bond in
non-deamidated human DNase and the Arg-73-Asp-74 peptide bond in the Asp
form of deamidated human DNase are cleaved by trypsin. Hence,
non-deamidated DNase is indicated in the tryptic map by the presence of T7
peptide shown in Table I, while the Asp-74 form of deamidated human DNase
is indicated in the tryptic map by the presence of the deamidated T7
peptide, called (D)T7. These three reporter peptides are labelled in FIG.
3. Unfortunately, trypsin only partially cleaves the peptide bond at the
C-terminal side of T7, between residues 77 and 78, so that each of the
reporter peptides T7, (D)T7 and T6-7 has a T8-conjugate, T7-8, (D)T7-8 and
T6-7-8, respectively. These six reporter peptides must therefore be
accounted for in order to quantitate deamidated human DNase by the tryptic
mapping method.
In principle, the (D)T7, (D)T7-8, T6-7 and T6-7-8 peptides represent
deamidated human DNase and the T7 and T7-8 peptides represent
non-deamidated human DNase and knowledge of the relative proportions of
these peptides permits a straightforward calculation of the extent of
deamidation in a preparation of DNase. In order to calculate the fraction
of the sample that is deamidated DNase, knowledge of the molar ratios of
deamidated and non-deamidated species is required, but the tryptic map
gives peak areas of individual peptides, not molar amounts. There are two
additional problems in the tryptic mapping procedure that must be
overcome: one chromatographic problem and one detection problem. The
chromatographic problem is that the T2 peptide coelutes with T6-7, and so
impedes the integration of an accurate peak area of this
deamidation-indicating peptide. This problem can be overcome by
integration of the chromatogram obtained at 280 nm, since all six of the
relevant peptides have at least one tyrosine (Tyr) residue, and so absorb
strongly at 280 nm, while T2 contains no Tyr or tryptophan (Trp) residues
and thus absorbs negligibly at this wavelength. The detection problem is
that the T6-7 and T6-7-8 peptides each contain three Tyr residues while
the other four peptides each contain only one. Thus the T6-containing
peptides have a higher molar absorptivity than do the peptides that
contain only T7, and a simple comparison of peak areas would tend to
overestimate the content of deamidated species in a sample. This problem
is overcome by normalizing the peak areas of the six peptides to the
number of Tyr residues in the peptide. Normalizing the peak areas in this
manner implies that all tyrosine residues in each of the peptides is in an
equivalent chemical environment, which is probably a good assumption for
relatively small peptides such as considered here. Upon normalization, the
corrected peak areas for deamidated and non-deamidated peptides can be
compared to arrive at an estimate of the content of deamidated DNase in a
sample.
2. Tentacle Cation Exchange Chromatography
Tentacle cation exchange (TCX) resins, unlike conventional cation exchange
resins, have polyionic ligands bound to a silica surface. The ligands of
the LiChrospher.RTM. 1000 SO.sub.3.sup.- column (EM Separations,
Gibbstown, N.J.) used in this example are advertised as containing between
25 and 50 sulfopropyl groups along a polyethylene backbone that is joined
at one end to the silica surface.
The TCX chromatogram of a sample of recombinant human DNase run on a
LiChrospher.RTM. 1000 SO.sub.3.sup.- column is shown in FIG. 5.
Recombinant human DNase was purified from cultures of Chinese hamster
ovary (CHO) cells transformed with DNA encoding human DNase. Shak, et al.,
Proc. Nat. Acad. Sci. 87:9188-9192 (1990); Shak, et al., International
Patent Application Publication No. WO 90/07572 (published 12 Jul. 1990).
The two peaks obtained were collected and subjected to several analyses in
order to identify them as the forms of DNase differing only at the residue
at amino acid position 74. FIG. 6 shows tryptic maps of the two peaks
collected from the TCX column, confirming that they are, respectively, the
deamidated and non-deamidated forms of human DNase. The tryptic map also
reveals that both forms of deamidated DNase (having Asp and iso-Asp at
amino acid position 74) are present in the first peak from the TCX
separation. Table II shows the specific activities measured for the two
peaks, confirming the relationship between deamidation and specific
activity inferred from the correlation shown in FIG. 2, and further
supporting the identification of the TCX fractions. Activity of the DNase
fraction was determined by a methyl green (MG) assay.
TABLE II
______________________________________
ACTIVITIES OF FRACTIONS COLLECTED FROM
TCX COLUMN.
MG and ELISA concentrations are the averages of
determinations on two samples.
MG ELISA Specific
Sample (.mu.g/ml)
(.mu.g/ml)
Activity
______________________________________
Starting preparation
8315 7828 1.06
of recombinant human
Dnase (load)
TCX Peak 1 85.3 119.7 0.71
(deamidated)
TCX Peak 2 (non-
149.2 99.4 1.50
deamidated)
______________________________________
A mutant form of human DNase, having an Asp residue at amino acid position
74, was produced by site-directed mutagenesis of the DNA encoding native
mature human DNase. This mutant coelutes with the first peak obtained in
the above chromatography, as shown in FIG. 7.
The following is the procedure used to pack the LiChrospher.RTM. 1000
SO.sub.3.sup.- tentacle cation exchange resin. Another tentacle cation
exchange resin similarly useful for separation of deamidated and
non-deamidated forms of human DNase is Fractogel.RTM. tentacle cation
exchange resin (EM Separations, Gibbstown, N.J.). LiChrospher and
Fractogel are registered trademarks of EM Industries, Inc., Hawthorne,
N.Y., or E. Merck, Darmstadt, West Germany. The "strong" forms of the
tentacle cation exchange resins (whether LiChrospher or Fractogel), having
a SO.sub.3.sup.- functional group, appear at this time to give the best
results.
3. HPLC Column Packing Procedure for LiChrospher.RTM.1000 SO.sub.3.sup.-
Resin
a. Materials and Equipment
1. Superformance glass cartridge 1.0 cm.times.5.0 cm bed.
2. Packing Buffer: 10mM sodium acetate, 1 mM CaCl.sub.2, pH to 4.5 with
acetic acid. Filter through a 0.2.mu. filter.
3. Column packing reservoir with a capacity of 20 ml. (Alltech part # 9501
or equivalent).
4. Empty 4.6 mm.times.50 mm stainless steel column with 0.5.mu. cut-off
frits
5. HPLC pump capable of maintaining a back pressure of 2000 psi (Waters
Model 510 or equivalent).
b. Packing Procedure.
1. De-fine resin:
a) Unpack 1.0 cm.times.5.0 cm Superformance glass column (Bed volume=3.93
ml resin). Resuspend resin to 20 mls in a clear glass, capped vessel with
column packing buffer. Slurry into a uniform suspension and divide into
2.times.10 ml aliquots. Add 10 mls of column packing buffer to each
aliquot to achieve suspensions of approx. 1.95 mls resin in 20 mls packing
buffer.
b) Slurry resin to achieve a uniform suspension. Allow to settle until
particles form a solid bed on the bottom of the vessel (2-4 hours).
Carefully pour off the supernatant containing fine particles.
c) Add 20 mls. packing buffer to resin and repeat step b). This procedure
should be repeated at least four times to assure removal of all fine resin
particles.
2. Column Packing:
a) Connect 4.6 mm.times.50 mm empty HPLC column to packing reservoir.
Slurry resin in 20 mls of packing buffer.
b) Add slurried resin to reservoir and quickly cap. Pump packing buffer at
a pressure that does not exceed 2000 psi. Adjust flow rate so that packing
pressure remains constant at about 2000 psi and flow for 15 minutes after
pressure stabilizes. Remove column and attach top end. Column may be used
directly or stored in 0.02% sodium azide.
For most samples, including DNase formulated in 150 mM NaCl, no sample
preparation is required prior to injection of the sample onto the column.
The column is equilibrated with a pH 4.5 acetate buffer containing calcium
ions, the sample is injected, and the column then is eluted with a salt
gradient. The following procedure is useful for small-scale separations of
deamidated and non-deamidated forms of human DNase. The proportions of the
peak areas on the resulting chromatogram are equal to the proportions of
deamidated and non-deamidated DNase in the sample.
Step 1. Load sample, containing up to 150 mM NaCl and at a pH up to 9 into
autosampler vial.
Harvested cell culture fluid samples require adjustment of pH to 4.5 and
centrifugation to remove proteins that are insoluble in the buffers used
in this procedure.
Step 2. Separate the two forms of DNase by HPLC under the following
conditions:
Column: TCX LiChrospher.RTM. 1000 SO.sub.3.sup.- repacked into a steel
column. Column dimensions of 4.6.times.50 mm and 4.6.times.150 mm have
been packed and employed.
Column temperature: ambient.
Eluent A: 10 mM sodium acetate, 1 mM
CaCl12, pH 4.5.
Eluent B: 1M NaCl in buffer A.
Gradient profile:
______________________________________
Time (min) % A % B
______________________________________
0 100 0
4 100 0
30 30 70
30.1 5 95
37 5 95
______________________________________
Flow rate: 0.8 ml/min (50 mm column), 0.5 ml/min (150 mm column).
Sample injection volume: up to 250 .mu.l.
Post-run column reequilibration time at
100% A: 20 min.
Autosampler compartment temperature: 5.degree..
Detection: Absorbance at 280 nm.
Step 3. Integrate chromatogram. Calculate the proportion of deamidated
species based on the peak area of the earlier eluting deamidated DNase
relative to the total peak area of both forms.
Tentacle cation exchange chromatography also provides a means for
separating, at large scale, the deamidated and non-deamidated forms of
human DNase. Large scale separations are more conveniently carried out
using simplified elution operating conditions than are described above for
small-scale analytical separations of the two forms of DNase. Hence,
larger scale separations have been carried out on the Fractogel-supported
tentacle cation exchanger according to the following pH-elution procedure:
Step 1. Pack 31.6 column (1.6 cm i.d..times.15.7 cm high) with Fractogel
EMD SO.sub.3 -650M tentacle cation exchange resin (EM Separations,
Gibbstown, N.J.).
Step 2. Diafilter DNase load with equilibration buffer (30 mM sodium
acetate (NaAc), 1 mM calcium chloride (CaCl.sub.2), 50 mM sodium chloride
(NaCl), pH 5). Concentrate by ultrafiltration to volume of 355 mls and
concentration of 2.5 mg/ml.
Step 3. Wash column with 2.5 column volumes (CV) of 2% sodium hydroxide
(NaOH).
Step 4. Wash column with 2.5 CV of pre-equilibration buffer (300 mM NaAc,
1M NaCl, pH 5).
Step 5. Wash column with 2.5 CV of equilibration buffer.
Step 6. Load column with 1-1.3 g of diafiltered/ultrafiltered DNase (from
Step 2). Begin collecting fractions of column effluent upon commencement
of DNase load.
Step 7. Wash column with 5 CV of equilibration buffer.
Step 8. Wash column with 5 CV of pH 5.3 wash buffer (25 mM succinate, 1 mM
CaCl.sub.2, pH 5.3).
Step 9. Wash column with 10 CV of pH 5.4 wash buffer (25 mM succinate, 1 mM
CaCl.sub.2, pH 5.4).
Step 10. Wash column with 10 CV of pH 6 wash buffer (25 mM MES, 1 mM
CaCl.sub.2, pH 6.0).
Step 11. Combine fractions collected during Steps 6-8 to make a pool
consisting predominantly of deamidated DNase. Combine fractions collected
during Step 10 to make a non-deamidated DNase pool. Fractions collected
during Step 9 contain a mixture of the two forms of DNase and may be
recycled.
The protocol described above is one example of the use of a tentacle cation
exchange resin for a preparative purification of the two forms of
recombinant human DNase in a manner that is scaleable to large-scale
recovery of purified deamidated and purified non-deamidated DNase.
4. Heparin and Immobilized DNA Analog Chromatography
In FIG. 8 chromatograms are aligned of analyses on a TSK Heparin column
(Toso Haas, Montgomeryville, Pa.) of samples containing either a mixture
of deamidated and non-deamidated forms of human DNase, purified deamidated
human DNase, or purified non-deamidated human DNase. The TSK-Heparin
column was run under the same conditions as described above for running
the analytical TCX column. The aligned chromatograms demonstrate that the
column of immobilized heparin resolves deamidated and non-deamidated forms
of DNase.
As described above, another means of separating the deamidated and
non-deamidated forms of DNase is to employ a column containing an
immobilized analog of DNA that is resistant to hydrolysis by DNase. One
example of this approach to an immobilized DNA analog column involved the
synthesis of the phosphorothioate oligonucleotide
5'--GCGCGCGCGCGCGCGCGCGCGC-NH.sub.3 -3'. This self-complementary sequence
can be annealed into a double-stranded form, and coupled to a Rainin
Hydropore-EP column (Rainin Co., Woburn, Mass.). FIG. 9 shows aligned
chromatograms of the analyses on this column of samples containing either
a mixture of deamidated and non-deamidated forms of human DNase, purified
deamidated human DNase, purified non-deamidated human DNase, or purified
mutant human DNase having an aspartic acid residue (rather than an
asparagine residue) at amino acid position 74. The column was run for
these analyses in a buffer containing 1 mM calcium chloride, 5 mM MES at a
pH of 6, and eluted with a linear gradient in salt concentration to 1M
sodium chloride over 20 minutes at a flow rate of 1 ml/min. As shown in
FIG. 9, under these conditions deamidated and non-deamidated DNase forms
are partially separated from each other. In addition, the two isomeric
forms of deamidated DNase, that differ at amino acid position 74 of the
DNase sequence by having either aspartic acid or iso-aspartic acid at this
position, are also resolved by this column. Thus an additional benefit of
this chromatographic method is that it allows the isolation of the two
isomers that arise on deamidation of human DNase.
5. Enzymatic Activity of Deamidated Human DNase and Non-deamidated Human
DNase
Several analytical methods have been used to examine the effect of
deamidation on the enzymatic activity of human DNase. Purified deamidated
human DNase and purified non-deamidated human DNase for use in these
studies were prepared by TCX chromatography, as described above.
In one method for determination of DNase enzymatic activity, synthetic
double stranded DNA, 25 base pairs in length, was labeled with
dinitrophenol (DNP) on one end and with biotin on the other end.
Hydrolysis of the substrate by DNase was detected by capture of the
reaction products on microtiter plate wells coated with antibody to DNP
and by quantitation of the intact probe with streptavidin-horseradish
peroxidase. The specific activity of stability samples was correlated
(r.sup.2 =0.613;n=5) with the extent of DNase deamidation (range 27%-93%).
Extrapolation of the least squares linear equation provided an estimate
that the specific activity of deamidated human DNase was approximately 77%
lower than that of non-deamidated human DNase.
Another method for determination of DNase enzymatic activity involved
hydrolysis of the chromogenic substrate p-nitrophenyl phenylphosphonate
(PNPP) as described by Liao, et al., Biochem. J. 255:781-787 (1988). The
kinetics of PNPP hydrolysis by human DNase are sigmoidal and were fit to
the Hill equation by nonlinear regression. By this method the V.sub.max of
fully deamidated human DNase was determined to be 77% lower than that of
non-deamidated human DNase. The substrate concentration for half maximal
activity (S.sub.0.5) did not differ significantly for the deamidated and
non-deamidated human DNase samples.
Another method for determination of DNase enzymatic activity is the assay
described by Kunitz, J. Gen. Physiol. 33:349 (1950), preferably modified
such that the enzymatic reaction is carried out at about pH 7.0-7.5. By
this method, the enzymatic activity of deamidated human DNase also was
determined to be lower than that of non-deamidated human DNase.
6. In Vitro Storage of Human DNase
Human DNase purified from recombinant CHO cells was dissolved at a
concentration of 4 mg/ ml in an unbuffered aqueous solution of 150 mM NaCl
and 1 mM CaCl.sub.2. Samples of the resulting DNase solution were then
placed into glass and plastic vials. Two different types of plastic vials
were used, one being made of Dupont 20 plastic resin (manufactured by E.I.
du Pont de Nemours & Co., Inc., Wilmington, Del. U.S.A.), and the other
being made of Escorene plastic resin (manufactured by Exxon Corp.). Both
of those plastics are low density polyethylene, but containers formulated
with other plastics, such as polypropylene, polystyrene, or other
polyolefins also may be used. The vials containing the DNase solution were
stored at either -70.degree. C., 2.degree.-8.degree. C., or 25.degree. C.
Initially, about 60%-65% of the DNase in the solutions was deamidated.
The DNase solutions in the vials were assayed at several times after
initial storage to determine the extent of deamidation of the DNase. The
results of those assays are shown in Table III.
TABLE III
______________________________________
% DEAMIDATION OF RECOMBINANT HUMAN
DNASE STORED IN GLASS AND PLASTIC VIALS.
Sample Day -70.degree. C.
2-8.degree. C.
25.degree. C.
______________________________________
Glass 83 66 66 78
174 63 66 81
Dupont 20 83 65 66 71
174 63 63 70
Escorene 83 65 66 71
174 64 62 70
______________________________________
After 83 and 174 days storage at -70.degree. C. or 2.degree.-8.degree. C.,
no difference was found in the amount of deamidated DNase in the plastic
vials and the amount of deamidated DNase in the glass vials. In each such
case, approximately 64% (+/-2%) of the DNase in the vials was deamidated
DNase.
Unexpectedly, however, after 83 or 174 days storage at 25.degree. C., there
was a difference in the amount of deamidated DNase in the plastic vials
and the amount of deamidated DNase in the glass vials. Significantly less
deamidated DNase was present in the plastic vials. In particular, after 83
days storage at 25.degree. C., 78% of the DNase in the glass vials was
deamidated DNase, whereas only about 70% of the DNase in the plastic vials
was deamidated DNase. After 174 days storage at 25.degree. C., 81% of the
DNase in the glass vials was deamidated DNase, whereas only about 71% of
the DNase in the plastic vials was deamidated DNase.
Without limiting the invention to any particular mechanism or theory of
operation, it may be that the differences in deamidation of DNase in
plastic and glass vials may be a consequence of differences in the pH of
the solutions in the vials. Initially, the pH of the DNase solution in the
glass vials was slightly higher than that in the plastic vials
(approximately pH 6.7 and approximately pH 6.5, respectively). The pH of
the DNase solution in the glass vials continued to increase slightly over
time (to approximately pH 6.9 after 83 days storage at 25.degree. C., and
approximately pH 7.0 after 174 days storage at 25.degree. C.), perhaps as
consequence of silicates or ions from the glass surface dissolving in the
solution. At higher pH, the rate of deamidation of human DNase is
increased. Since it was not appreciated that deamidation of human DNase
occurs at elevated pH, it is an embodiment of this invention to formulate
and/or store human DNase in solutions having acidic pH, typically at about
pH 4.5-6.8 and most preferably at about pH 5.0-6.8.
Thus, a significant improvement in the stability of human DNase in solution
is obtained by placing such DNase solution in plastic vials rather than
glass vials, with apparently less deamidation of the DNase occurring over
time in the plastic vials than in the glass vials. This finding may be
especially relevant to the choice of packaging of human DNase for
therapeutic use, where it is especially desirable that the human DNase be
capable of storage for extended periods of time without significant loss
of enzymatic activity. Of course, glass vials with non-glass coatings, for
example, plastic linings, would be equally useful. What is important is to
avoid storing DNase in contact with glass, especially for storage
exceeding about 15-30 days.
General Remarks
The foregoing description details specific methods which can be employed to
practice the present invention. Having detailed specific methods used to
identify, characterize, separate and use the pure deamidated and
non-deamidated human DNase hereof, and further disclosure as to specific
model systems pertaining thereto, those skilled in the art will well
enough know how to devise alternative reliable methods for arriving at the
same information in using the fruits of the present invention. Thus,
however detailed the forgoing may appear in text, it should not be
construed as limiting the overall scope hereof; rather, the ambit of the
present invention is to be determined only by the lawful construction of
the appended claims.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 17
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 346 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
SerCysThrGlySerAlaLeuLysCysPh ePheArgAspLeuSer
151015
SerXaaThrThrPhePheSerLeuSerSerLysArgArgLysLeu
2025 30
SerSerLysAspIleProAspSerXaaGlnHisSerArgHisLeu
354045
XaaGlyHisHisHisHisLeuArgMetArgGlyMetLysLeuLeu
505560
GlyAlaLeuLeuAlaLeuAlaAlaLeuLeuGlnGlyAlaValSer
657075
LeuLysIleA laAlaPheAsnIleGlnThrPheGlyGluThrLys
808590
MetSerAsnAlaThrLeuValSerTyrIleValGlnIleLeuSer
95 100105
ArgTyrAspIleAlaLeuValGlnGluValArgAspSerHisLeu
110115120
ThrAlaValGlyLysLeuLeuAspAsnLe uAsnGlnAspAlaPro
125130135
AspThrTyrHisTyrValValSerGluProLeuGlyArgAsnSer
140145 150
TyrLysGluArgTyrLeuPheValTyrArgProAspGlnValSer
155160165
AlaValAspSerTyrTyrTyrAspAspGlyCysGluProCysGly
170175180
AsnAspThrPheAsnArgGluProAlaIleValArgPhePheSer
185190195
ArgPheThrG luValArgGluPheAlaIleValProLeuHisAla
200205210
AlaProGlyAspAlaValAlaGluIleAspAlaLeuTyrAspVal
215 220225
TyrLeuAspValGlnGluLysTrpGlyLeuGluAspValMetLeu
230235240
MetGlyAspPheAsnAlaGlyCysSerTy rValArgProSerGln
245250255
TrpSerSerIleArgLeuTrpThrSerProThrPheGlnTrpLeu
260265 270
IleProAspSerAlaAspThrThrAlaThrProThrHisCysAla
275280285
TyrAspArgIleValValAlaGlyMetLeuLeuArgGlyAlaVal
290295300
ValProAspSerAlaLeuProPheAsnPheGlnAlaAlaTyrGly
305310315
LeuSerAspG lnLeuAlaGlnAlaIleSerAspHisTyrProVal
320325330
GluValMetLeuLysXaaAlaAlaProProHisThrSerXaaThr
335 340345
Ala
346
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1039 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
TCCTGCACAGGCAGTGCCTTGAAGTGCTTCTTCAG AGACCTTTCTTCATA50
GACTACTTTTTTTTCTTTAAGCAGCAAAAGGAGAAAATTGTCATCAAAGG100
ATATTCCAGATTCTTGACAGCATTCTCGTCATCTCTGAGGACATCACCAT150
CATCTCAGGATGAGGGGCATGAAGCTGCTGGGGGCGCTGCTGGCACTGG C200
GGCCCTACTGCAGGGGGCCGTGTCCCTGAAGATCGCAGCCTTCAACATCC250
AGACATTTGGGGAGACCAAGATGTCCAATGCCACCCTCGTCAGCTACATT300
GTGCAGATCCTGAGCCGCTATGACATCGCCCTGGTCCAGGAGGTCAGAGA350
CAGCC ACCTGACTGCCGTGGGGAAGCTGCTGGACAACCTCAATCAGGATG400
CACCAGACACCTATCACTACGTGGTCAGTGAGCCACTGGGACGGAACAGC450
TATAAGGAGCGCTACCTGTTCGTGTACAGGCCTGACCAGGTGTCTGCGGT500
GGACAGCTACTACTACGA TGATGGCTGCGAGCCCTGCGGGAACGACACCT550
TCAACCGAGAGCCAGCCATTGTCAGGTTCTTCTCCCGGTTCACAGAGGTC600
AGGGAGTTTGCCATTGTTCCCCTGCATGCGGCCCCGGGGGACGCAGTAGC650
CGAGATCGACGCTCTCTATGACGTCTACCT GGATGTCCAAGAGAAATGGG700
GCTTGGAGGACGTCATGTTGATGGGCGACTTCAATGCGGGCTGCAGCTAT750
GTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGACAAGCCCCACCTT800
CCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACACCC ACGCACT850
GTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTT900
GTTCCCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCTATGGCCTGAG950
TGACCAACTGGCCCAAGCCATCAGTGACCACTATCCAGTGGAGGTGATGC1000
TGAAGTGAGCAGCCCCTCCCCACACCAGTTGAACTGCAG1039
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
IleAlaAlaPheAsnIleGlnThrPheGlyGluThrLys
151013
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
MetSerAsnAlaThrLeuValSerTyrIleValGln IleLeuSer
151015
Arg
16
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
TyrAspIleAl aLeuValGlnGluValArg
1510
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
AspSerHisLeuThrAlaValGlyLy s
159
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
LeuLeuAspAsnLeuAsnGlnAspAlaProAspThrTyrHisTyr
151015
ValValSerGluProLeuGlyArg
2023
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
AsnSerTyrLys
14
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
TyrLeuPheValTyrArgProAspGlnValS erAlaValAspSer
151015
TyrTyrTyrAspAspGlyCysGluProCysGlyAsnAspThrPhe
2025 30
AsnArg
32
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GluProAlaIleValArg
156
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
PhePheSerArg
14
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
PheThrGluValArg
15
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GluPheAlaIleValProLeuHisAlaAlaProGlyAspAla Val
151015
AlaGluIleAspAlaLeuTyrAspValTyrLeuAspValGlnGlu
202530
Lys
31
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
TrpGlyLeuGluAspValMetLeuMetGlyAspPheAsnAlaGly
15 1015
CysSerTyrValArgProSerGlnTrpSerSerIleArg
202528
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
LeuTrpThrSerProThrPheGlnTrpLeuIleProAspSerAla
151015
AspThrThrAlaThrProThrHisCysAlaTy rAspArg
202528
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
IleValValAlaGlyMetLeuLeuArg
159
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GlyAlaValValProAspSerAlaLeuProPheAsnPheGlnAla
1 51015
AlaTyrGlyLeuSerAspGlnLeuAlaGlnAlaIleSerAspHis
202530
TyrProValGlu ValMetLeuLys
3538
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